This invention relates to a method of culturing microbes which belong to the genus Bordetella and a culture medium to be used in the culture and a method for preparing biologically active substances using said culture medium.
The microbes of the genus Bordetella include Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, etc., of which Bordetella pertussis is especially known as a pathogenic bacterium which attacks the tracheas, bronchia, and bronchiolus of an infected patient to cause whooping cough and aflicts the patient with a paroxysmal cough for a long duration of several weeks. A means of prompt and accurate detection of the Bordetella pertussis, etc. is therefore earnestly desired from a clinical viewpoint. Such a desired method of cultivation or a culture medium should promote the culture of clinically obtained and separated causative bacteria. Also, such a method of cultivation or the culture medium should promote the growth of Bordetella pertussis for the efficient production of pertussis vaccine (killed whole cell vaccine or component vaccine) which is used for the prevention of whooping cough. Medically effective biologically active substances such as islet activating protein (hereinafter referred to as IAP) from which a remedy and prevention of diabetes is expected to be developed, leukocytosis promoting factor-hemagglutinin (hereinafter referred to as LPF-HA) and filamentous-hemagglutinin (hereinafter referred to as F-HA) which are attracting medical attention as acellular pertussis vaccine component, etc. are obtained from the cultured substance (consisting of the culture medium and cultured bacteria) of Bordetella pertussis Phase I. An effective method for culturing Bordetella pertussis is sought in order to achieve an increased productivity in the preparation of the above-described biologically active substances.
As for the culture medium for the growth of bacteria belonging to the genus Bordetella, especially Bordetella pertussis phase I, there are well-known culture media, in which active carbon and starch or ion-exchange resin are involved, originated by Cohen et al. (American Journal of Public Health, vol 36, pp. 371-376, 1946) and Sutherland et al. (Journal of Pathology and Bacteriology, vol. 82, pp. 431-438, 1961). However, charcol and ion-exchange resin which can't permit uniformity in the culture medium restrict the work of monitoring the proliferation of bacteria and also the effect on eliminating the preventive factors such as fatty acid, etc. which arrest the growth of bacteria is not always selective because of their unspecific absorbability. Furthermore, the effect of starch upon the growth of bacteria is not sufficient. It has therefore been desired to develop a new culture medium in which the above-mentioned disadvantages are all corrected. In order to clinically isolate Bordetella pertussis as exclusive single colonies, it is necessary to make airborne droplets emitted from an infected patient directly contact a solid agar culture medium such as Bordet-Gengou medium (hereinafter referred to as BG medium). However, a culture medium of this kind requires fresh blood as an essential component, therefore, its preservability is very low, and possesses a defect of being not stable of colony-forming ability. From the points of view mentioned above, the development of an culture medium for clinical isolation having a certain clearly defined chemical composition and high preservability has long been hoped for.
In recent years, a synthetic culture medium has been developed by Stainer and Scholte for mass cultivation of Bordetella pertussis (Journal of General Microbiology, vol. 63, pp. 211-220, 1971). Since Stainer-Scholte medium (hereinafter referred to as SS medium) does not contain such additives as blood and polypeptone arising from natural sources that are inducible of qualitative difference between lots, it is possible to achieve a strict control of the medium composition and carry out the cultivation of bacteria without bringing about changes in their nature. Besides, it has another merit of being capable of keeping away other unnecessary reactogenic proteins in the culture system during the processes of separating and purifying biologically active substances such as before-mentioned IAP and LPF-HA. Because of the characteristics mentioned above, SS medium has recently come to be widely used in commerically preparing pertussis vaccine and biologic active substances from Bordetella pertussis; however, it has the disadvantage of not promissing enough production of LPF-HA in the liquid culture under shaking or static conditions and also not promissing stable growth of bacteria when the inoculation is conducted at seed concentration of 10.sup.7 cells/ml or less. Solid SS agar medium (hereinafter referred to as SSA medium) obtained by solidifying SS medium by use of agar amounting to 1 to 2% of the whole has an evident disadvantage of not being capable of forming colonies when inoculating at a low concentration of 10.sup.7 cells/ml or less.